Peripheralization of hematopoietic stem cells

ABSTRACT

The invention relates to in vivo peripheralization of CD34 +  cells by administering anti-VLA-4 antibodies or anti-VCAM-1 antibodies.

This application is a national stage application under U.S.C. 371 ofPCT/US93/11060, filed Nov. 15, 1993, which is a continuation-in-part ofSer. No. 07/977,702, filed Nov. 13, 1992, now abandoned.

FIELD OF THE INVENTION

The invention relates to the manipulation of hematopoietic stem cells.More particularly, the invention relates to methods for increasing thenumber of hematopoietic stem cells in peripheral blood.

BACKGROUND OF THE INVENTION

Hematopoietic stem cells are primitive, uncommitted progenitor cellsthat give rise to the lymphoid, myeloid and erythroid lineages of cellsin blood. The stem cell population constitutes only a small proportionof the total cells in bone marrow and represents even a far moreminuscule proportion of the cells in peripheral blood.

Stem cells have commonly been characterized by their surface antigenicdeterminants. Tsukamoto et al., U.S. Pat. No. 5,061,620 (1991), teachesthat a highly stem cell concentrated cell composition is CD34⁺, CD10⁻,CD19⁻ and CD33⁻. Leon et al., Blood 77:1218-1227 (1991), teaches thatabout one per cent of CD34⁺ cells, or about 0.01% of the total marrowcell population, do not express differentiation antigens, such as CD33(myeloid lineage), CD71 (erythroid lineage) or CD10 and CD5 (lymphoid Band T lineage), and that reduced expression of CD34 expression duringmaturation is associated with increased expression of thedifferentiation antigens.

Combinations of antigenic and functional characteristics have also beenused to characterize stem cells. Sutherland et al., Proc. Natl. Acad.Sci. U.S.A. 87:3584-3588 (1990), teaches that primitive stem cells donot bind to soybean agglutinin, express high levels of CD34, form blastcolonies with high plating efficiency and are enriched in long-termculture initiating cells (LTC-IC). Craig et al., Blood Reviews 6:59-67(1992), teaches that the CFU-GM assay is the most widely used measure ofthe hematopoietic progenitor viability of a bone marrow or peripheralblood stem cell harvest, and correlates well with per cent CD34⁺.Spangrude, Immunology Today 10:344-350 (1989), teaches that stem cellsaccumulate low levels of rhodamine 123 relative to other bone marrowcell types. Chaudhaury et al., Cell 66:85-94 (1991), teaches that stemcells express high levels of P-glycoprotein relative to other marrowcell types.

The ability to manipulate hematopoietic stem cells has becomeincreasingly important in the development of effective chemotherapeuticand radiotherapeutic approaches to the treatment of cancer. Currentapproaches to chemotherapy and radiotherapy utilize bone marrowtransplantation (BMT). BMT involves removing one to two liters of viablepelvic bone marrow containing stem cells, progenitor cells and moremature blood cells, treating the patient with chemotherapy orradiotherapy to kill tumor cells, and reintroducing bone marrow cellsintravenously. BMT, however, suffers from many disadvantages. Harvestingof BM for BMT requires general anaesthesia, which increases both riskand cost. In addition, if cancer cells are present in the marrow and arenot rigorously purged, recurrence of the disease is a distinct risk.Also, if widespread invasion of bone marrow by cancer cells (myeloma,Waldenstrom's macroglobulinemia) is present, peripheral blood cells arethe only option for use in autologous transplantation (ABMT). Finally,patients who have undergone pelvic irradiation are not candidates forABMT.

As a result of these difficulties, much interest has been developed inproviding methods for obtaining stem cells from peripheral blood forautologous supply of stem cells to patients undergoing chemotherapy.Autologous supply of stem cells from peripheral blood would allow theuse of greater doses of chemo- or radiotherapy, but with less risk thanBMT. In addition, the use of stem cells from peripheral blood does notrequire anaesthesia to obtain the stem cells. Also, Lowry, Exp. Hematol.20:937-942 (1992), teaches that cancer cells in the marrow tend not toperipheralize. The critical limitation in such a procedure, however,lies in the very small number of stem cells ordinarily present inperipheral blood. Lobo et al., Bone Marrow Transplantation 8:389-392(1991), teaches that addition of peripheral blood stem cells collectedin the absence of any peripheralization techniques does not hastenmarrow recovery following myeloablative therapy. In contrast, Haas etal., Exp. Hematol. 18:94-98 (1990), demonstrates successful autologoustransplantation of peripheral blood stem cells mobilized withrecombinant human granulocyte-macrophage colony-stimulating factor(GM-CSF). Thus, increasing the number of stem cells in peripheral bloodby peripheralization techniques is critical to the success of proceduresutilizing peripheral blood as a source for autologous stem celltransplantation. Other cytokines may be useful in this regard. Rowe andRapoport, J. Clin. Pharmacol. 32:486-501 (1992), suggests that inaddition to GM-CSF, other cytokines, including macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(G-CSF), erythropoietin, interleukins-1, -2, -3, -4 and -6, and variousinterferons and tumor necrosis factors have enormous potential.

Another approach to autologous transplantation is to purify stem cellsfrom peripheral blood using immunoaffinity techniques. These techniqueshold promise not only for autologous stem cell transplantation inconjunction with chemotherapy, but also for gene therapy, in whichpurified stem cells are necessary for genetic manipulation to correctdefective gene function, then reintroduced into the patient to supplythe missing function. However, Edgington, Biotechnology 10:1099-1106(1992), teaches that current procedures require three separate four hoursessions to process enough cells in the absence of peripheralization.DePalma, Genetic Engineering News, Vol. 12, May 1, 1992, teaches thatthis can be improved by treatment with G-CSF for peripheralization.

These studies underscore the importance of developing new methods toeffect the peripheralization of hematopoietic stem cells. Onepossibility is to search for new ways to release stem cells from thebone marrow environment into the periphery. Unfortunately, little isknown about the types of molecular interactions that hold hematopoieticstem cells in the marrow environment in vivo. Recently, some in vitrostudies have been undertaken to look at the role of integrins,fibronectin, and other surface antigens in binding between stem cellsand bone marrow stromal cells.

Integrins are a large family of integral membrane glycoproteins havingover 16 heterodimeric members that mediate interactions between cells,interactions between cells and the extracellular matrix, andinteractions involved in embryonic development and regulation of T-cellresponses. Among integrins, the VLA-5 (α⁵β₁) complex is widelydistributed and functions as a receptor for fibronectin. The VLA-4(α⁴β₁) complex is expressed at substantial levels on normal peripheralblood B and T cells, thymocytes, monocytes, and some melanoma cells anwell as on marrow blast cells and erythroblasts. Ligands for VLA-4 arevascular cell adhesion molecule-1(VCAM-1) and CS-1, an alternatelyspliced domain within the Hep II region of fibronectin. Another group ofintegrins (CDIIa/CD18, CDIIb/CD18, and CDIIc/CD18) share the common β₂chain and are variably expressed on peripheral T cells, monocytes, andmature granulocytes. Ligands for β₂-integrins include members of the Igsuperfamily (ICAM-1 and ICAM-2) found on activated endothelial cells.

Issekutz, J. Immunol. 147:4178-4184 (1991), discloses that TA-2, amonoclonal antibody to rat VLA-4, inhibits the in vivo migration, ofsmall peritoneal exudate lymphocytes and lymphocytes from peripherallymph nodes, from the blood across the vascular endothelium to sites ofinflammation. This document also observes that systemic treatment ofrats with TA-2 was accompanied by an increase in total blood lymphocytecount.

Taixido et al., J. Clin. Invest. 90:358-367 (1992), teaches that in anin vitro model, interactions between VLA-4/VCAM-1, VLA-5/fibronectin andβ₂-integrin/ICAM-1 are all important for adhesion between bone marrowstromal cells and cells expressing high levels of CD34. Simmons et al.,Blood 80:389-395 (1992), teaches that in an in vitro model, adhesionbetween stromal cells and CD34⁻ cells was predominantly dependent on theVLA-4/VCAM-1 interaction and was largely inhibited by monoclonalantibodies to either VLA-4 or VCAM-1, with fibronectin playing a minorrole in binding. Williams et al., Nature 352:438-441 (1991), using invivo mouse studies, teaches that adhesion of murine hematopoietic stemcells to stromal cell extracellular matrix (ECM) is partly promoted byproteolytic fragments of fibronectin containing an alternatively splicedregion of the IIICS domain, and suggest that the interaction is likelyto be mediated by VLA-4. All of these studies utilized antibodies toprevent adherence between stem cells and their microenvironment.However, none have analyzed whether such interactions are reversible, orperturbable after adherence has taken place. These results indicate theneed for further studies to determine what interactions between the bonemarrow environment and hematopoietic stem cells are responsible forkeeping the stem cells within that environment in vivo and whether suchinteractions can be perturbed to effect peripheralization of stem cells.

There is, therefore, a need for new methods for peripheralizing stemcells, both for scientific investigatory purposes for understanding theprocesses of peripheralization and homing, and for the development ofbetter methods of peripheralization for autologous stem celltransplantation in the course of cancer treatment or gene therapy.Preferably, such methods should produce even higher levels of stem cellsin peripheral blood than existing methods provide.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides a novel method for increasingthe number of hematopoietic stem cells and CD34⁺ cells in peripheralblood, which is also known as “peripheralization” or “mobilization” ofhematopoietic stem cells and CD34⁺ cells. This method comprises the stepof administering a blocking agent of VLA-4 antigens on the surface ofhematopoietic stem cells and CD34⁺ cells. Various agents can be used tomediate such blocking, including anti-VLA-4 or anti-VCAM-1 antibodieswhich may optionally be single chain, humanized or chimeric, Fab, Fab′,F(ab′)₂ or F(v) fragments thereof, heavy or light chain monomers ordimers thereof, or intermixtures of the same, soluble fibronectin, CS-1peptides or fibronectin peptides containing the amino acid sequenceEILDV or conservatively substituted amino acid sequences, or solubleVCAM-1, bifunctional VCAM-1/Ig fusion proteins or VCAM-1 peptides.

In another aspect, the invention provides a novel method forperipheralizing hematopoietic stem cells and CD34⁺ cells with morepredictable greater effectiveness than cytokine treatment aloneprovides. According to this aspect of the invention, the methodcomprises administering a blocking agent of VLA-4 antigens on thesurface of hematopoietic stem cells and CD34⁺ cells, as in the firstaspect of the invention, in combination with a stimulating agent ofhematopoietic stem cell proliferation. The step of administering astimulating agent of hematopoietic stem cell proliferation can becarried out by using a cytokine, preferably G-CSF, stem cell factor,totipotent stem cell factor, stem cell proliferation factor or GM-CSF,but alternatively M-CSF, erythropoietin, interleukins-1, -2, -3, -4, -6,or 11.

In another aspect, the invention provides an improved method oftransplanting peripheral blood stem cells into a patient who hasundergone chemotherapy or radiotherapy for cancer. In this method, priorto the administration of myeloablative chemotherapy or radiotherapy,stem cells are peripheralized from the patient's bone marrow byadministration of an agent that mediates blocking of VLA-4 antigens onthe surface of hematopoietic stem cells and CD34⁺ cells. This agent maybe administered alone, or preferably in conjunction with an agent thatstimulates proliferation of stem cells. The peripheralized stem cellsare then collected from peripheral blood by leukapheresis. Stem cellsare then enriched from the collected peripheralized blood byimmunoadsorption using anti-CD34 antibodies. Optionally, the enrichedstem cells are then expanded ex vivo by culturing them in the presenceof agents that stimulate proliferation of stem cells. Followingadministration of myeloablative chemotherapy or radiotherapy, theenriched, and optionally expanded stem cells are then returned to thepatient's circulating blood and allowed to engraft themselves into thebone marrow.

In another aspect, the invention provides an improved method oftransplanting peripheral blood stem cells into a patient who hasundergone myeloablative chemotherapy or radiotherapy for AIDS. Thismethod involves the same steps as described for transplantingperipheralized stem cells into a patient who has undergone chemotherapyor radiotherapy for cancer. In addition, this method further optionallyinvolves administration to the patient of anti-HIV agents, such asantivirals such as AZT, soluble CD4, and CD4-directed blockers of theAIDS virus or antisense or antigene oligonucleotides, both before andafter the return of the enriched and optionally expanded stem cells tothe patient's circulating blood. This step serves a “mopping up”function to prevent residual virus from infecting the progeny of thenewly returned stem cells.

In another aspect, the invention provides an improved method forcarrying out gene therapy in patients having various genetic andacquired diseases. In this method, stem cells are peripheralized fromthe patient's bone marrow by administration of an agent that mediatesblocking of VLA-4 antigens on the surface of hematopoietic stem cellsand CD34⁺ cells. As in the method previously described herein, thisagent may be administered alone or in conjunction with an agent thatstimulates proliferation of stem cells. Peripheral blood is thencollected by leukapheresis. Stem cells are then enriched from thecollected peripheral blood by immunoadsorption using anti-CD34antibodies. Optionally, the enriched stem cells are then expanded exvivo by culturing them in the presence of agents that stimulateproliferation of stem cells. The enriched and optionally expanded stemcells are then transduced with an amphotrophic retroviral vector, orother suitable vectors, that expresses a gene that ameliorates thegenetic or acquired disease. Optionally, the vector may also carry anexpressed selectable marker, in which case successfully transduced cellsmay be selected for the presence of the selectable marker. Thetransduced and optionally selected stem cells are then returned to thepatient's circulating blood and allowed to engraft themselves into thebone marrow.

It is an object of the invention to provide a method for peripheralizinghematopoietic stem cells and CD34⁺ cells as an experimental model forinvestigating hematopoiesis, homing of stem cells to the bone marrow,and cytokine-induced peripheralization of stem cells. It is a furtherobject of the invention to provide a method for optimizingperipheralization of hematopoietic stem cells and CD34⁺ cells to providestem cell-enriched peripheral blood for autologous transplantationfollowing chemo- or radiotherapy. It is a further object of theinvention to provide a method for peripheralizing CD34⁺ cells tomaximize the yield of purified hematopoietic stem cells and progenitorcells from peripheral blood, either for autologous transplantation ofthe stem cells following chemo- or radiotherapy, or for use in genetherapy. It is a further object of the invention to provide a method forperipheralizing stem cells and CD34⁺ cells without risk of causingcytokine-induced cell differentiation of normal stem cells orproliferation of contaminating leukemia cells. It is a further object ofthe invention to provide a peripheralization technique that haspredictable timing for the peak of progenitor content in peripheralblood for scheduling leukapheresis.

The invention satisfies each of these objects by providing a method forperipheralizing stem cells and CD34⁺ cells by administering a blockingagent of VLA-4 antigen on the surface of hematopoietic stem cells. Thiseffect can be increased by the use of such blocking agents inconjunction with approaches to amplify stem cells to produce asynergistic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following Figures, which further exemplify the claimed invention,dashed lines represent total white blood cell counts, as recorded on theright vertical axes. Black boxes represent BFUe as represented on theleft vertical axes. Downward arrows represent points of administrationof antibody. Horizontal axes represent days before and after firstadministration of antibody.

FIG. 1A is a profile of total white blood cells and CFU in peripheralblood before treatment of macaques.

FIG. 1B is a profile of total white blood cells and CFU in peripheralblood of a baboon after treatment with anti-VLA-4 antibodies.

FIG. 1C is a profile of total white blood cells and CFU in peripheralblood of a macaque after treatment with anti-VLA-4 antibodies.

FIG. 2 is a profile of total white blood cells and CFU in peripheralblood before treatment of an animal with the anti-CD18 monoclonalantibody 60.3. All symbols are as defined above.

FIG. 3A is a profile of the results of combined treatment with G-CSF andanti-VLA-4 monoclonal antibody HP1/2. The symbols are as describedabove, except that narrow downward-pointing arrows represent points ofG-CSF administration, bold downward-pointing arrows represent points ofantibody administration, and dotted line (with triangles) representtotal lymphocyte counts.

FIG. 3B is a profile of the results for a control animal treated withGCSF alone.

FIG. 4A shows high proliferative potential (HPP) progenitors (coloniesover 0.5 mm in diameter of compact growth) resulting from combinedtreatment with GCSF and HP1/2 antibody.

FIG. 4B is a profile of HPP progenitors resulting from treatment withGCSF alone. Symbols are as in FIG. 3.

FIGS. 5A and 5B are the nucleotide sequences encoding the variable heavyregion of the heavy and light chains of anti-VLA-4 murine monoclonalantibody HP 1/2.

FIG. 6A is a profile of the results of combined treatment with5-fluorouracil and anti-VLA-4 murine monoclonal antibody HP1/2. Symbolsare as described for FIG. 3.

FIG. 6B is a profile of the results of 5-fluorouracil treatment alone.

FIG. 7A is the nucleotide sequences of the ^(V) _(H)-encoding regionshaving CDR-encoding sequences from murine HP1/2 transplanted therein(SEQ ID NO:3).

FIG. 7B is the nucleotide sequence of the transplanted ^(V) _(K)sequence (SEQ ID NO:4).

FIG. 8A is a nucleotide sequences encoding the variable regions of theheavy and light chains of the humanized anti-VLA-4 antibody hHP1/2encoding the ^(V) _(H) region (SEQ ID NO:5).

FIG. 8B is the nucleotide sequence encoding the ^(V) _(K) region (SEQ IDNO:6).

FIG. 9 is a profile of the results of treatment with humanizedanti-VLA-4 antibody hHP1/2. Symbols are as described for FIG. 3.

FIG. 10 is a profile of the results of treatment with murine Fabfragments of anti-VLA-4 antibody HP1/2. Symbols are as described forFIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the manipulation of hematopoietic stem cells.More particularly, the invention relates to the peripheralization ofhematopoietic stem cells and other CD34⁺ cells.

In a first aspect, this invention provides a method for peripheralizinghematopoietic stem cells and CD34⁺ cells, comprising the step ofadministering a blocking agent of VLA-4 antigens on the surface ofhematopoietic stem cells and CD34⁺ cells. For purposes of thisinvention, the term “blocking agent of VLA-4 antigens” is intended tomean an agent that is capable of interfering with interactions betweenVLA-4 antigens and either VCAM-1 or fibronectin on the surface ofstromal cells or in the extracellular matrix (ECM). As demonstratedherein, such blocking of VLA-4 antigens causes peripheralization of stemcells and CD34⁺ cells. This demonstration utilized a monoclonal antibodyagainst VLA-4 as a blocking agent. Those skilled in the art willrecognize that, given this demonstration, any agent that can block VLA-4antigens can be successfully used in the method of this invention. Thus,for purposes of this invention, any agent capable of blocking VLA-4antigens on the surface of hematopoietic stem cells is considered to bean equivalent of the monoclonal antibody used in the examples herein.For example, this invention contemplates as equivalents at leastpeptides, peptide mimetics, carbohydrates and small molecules capable ofblocking VLA-4 antigens on the surface of CD34⁺ cells or hematopoieticstem cells.

In a preferred embodiment, the blocking agent that is used in the methodof this invention to block VLA-4 antigens on the surface ofhematopoietic stem cells and CD34⁺ cells is a monoclonal antibody orantibody derivative. Preferred antibody derivatives include humanizedantibodies, chimeric antibodies, single chain antibodies, Fab, Fab′,F(ab′)₂ and F(v) antibody fragments, and monomers or dimers of antibodyheavy or light chains or intermixtures thereof. The successful use ofmonoclonal antibody OKT3 to control allograft rejection indicates that,although humanized antibodies are preferable, murine monoclonalantibodies can be effective in therapeutic applications. Monoclonalantibodies against VLA-4 are a preferred blocking agent in the methodaccording to this invention. Human monoclonal antibodies against VLA-4are another preferred blocking agent in the method according to theinvention. These can be prepared using in vitro-primed humansplenocytes, as described by Boerner et al., J. Immunol. 147:86-95(1991). Alternatively, they can be prepared by repertoire cloning asdescribed by Persson et al., Proc. Natl. Acad. Sci. U.S.A. 88:2432-2436(1991) or by Huang and Stollar, J. of Immunol. Methods 141:227-236(1991). Another preferred blocking agent in the method of the presentinvention is a chimeric antibody having anti-VLA-4 specificity and ahuman antibody constant region. These preferred blocking agents can beprepared according to art-recognized techniques, as exemplified in U.S.Pat. No. 4,816,397 and in Morrison et al., Proc. Natl. Acad. Sci. U.S.A.81:6851-6855 (1984). Yet another preferred blocking agent in the methodof this invention is a humanized antibody having anti-VLA-4 specificity.Humanized antibodies can be prepared according to art-recognizedtechniques, as exemplified in Jones et al., Nature 321:522 (1986);Riechmann, Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci.U.S.A. 86:10029 (1989); and Orlandi et al., Proc. Natl. Acad. Sci.U.S.A. 86:3833 (1989). Those skilled in the art will be able to produceall of these preferred blocking agents, based upon the nucleotidesequence encoding the heavy and light chain variable regions of HP1/2[SEQ. ID. NOS. 1 and 2], as shown in FIG. 5, using only well knownmethods of cloning, mutagenesis and expression (for expression ofantibodies, see, e.g., Boss et al., U.S. Pat. No. 4,923,805). Two otherpreferred blocking agents are single chain antibodies, which can beprepared as described in U.S. Pat. No. 4,946,778, the teachings of whichare hereby incorporated by reference; and biosynthetic antibody bindingsites, which can be prepared as described in U.S. Pat. No. 5,091,513,the teachings of which are hereby incorporated by reference. Thoseskilled in the art will recognize that any of the above-identifiedantibody or antibody derivative blocking agents can also act in themethod of the present invention by binding the receptor for VLA-4, thusacting as agents for blocking the VLA-4 antigen on the surface ofhematopoietic stem cells, within the meaning of this term for purposesof this invention. Thus, antibody and antibody derivative blockingagents according to this invention, as described above, includeembodiments having binding specificity for VCAM-1 or fibronectin, sincethese molecules appear to either be important in the adhesion betweenstem cells and stromal cells or the extracellular matrix or interferewith traffic of stem cells through other tissues and blood.

In another preferred embodiment, the blocking agents used in the methodaccording to this invention are not antibodies or antibody derivatives,but rather are soluble forms of the natural binding proteins for VLA-4.These blocking agents include soluble VCAM-1, bifunctional VCAM-1/Igfusion proteins, or VCAM-1 peptides as well as fibronectin, fibronectinhaving an alternatively spliced non-type III connecting segment andfibronectin peptides containing the amino acid sequence EILDV or asimilar conservatively substituted amino acid sequence. These blockingagents will act by competing with the stromal cell- or ECM-bound bindingprotein for VLA-4 on the surface of stem cells.

In this method according to the first aspect of the present invention,blocking agents are preferably administered parenterally. The blockingagents are preferably administered as a sterile pharmaceuticalcomposition containing a pharmaceutically acceptable carrier, which maybe any of the numerous well known carriers, such as water, saline,phosphate buffered saline, dextrose, glycerol, ethanol, and the like, orcombinations thereof. Preferably, the blocking agent, if an antibody orantibody derivative, will be administered at a dose between about 0.1mg/kg body weight/day and about 10 mg/kg body weight/day. Fornon-antibody or antibody derivative blocking agents, the dose rangeshould preferably be between molar equivalent amounts to these amountsof antibody. Optimization of dosages can be determined by administrationof the blocking agents, followed by CFU-GM assay of peripheral blood, orassay of CD34⁺ cells in peripheral blood. The preferred dosage shouldproduce an increase of at least 10-fold in the CFU-GM counts inperipheral blood.

In a second aspect, the present invention provides a method forperipheralizing hematopoietic stem cells that is far more effective thancytokine treatment alone. According to this aspect of the invention, themethod comprises the step of administering a blocking agent of VLA-4antigens on the surface of hematopoietic stem cells in combination withthe step of administering a stimulating agent of hematopoietic stem cellproliferation in vivo. The step of administering a blocking agent ofVLA-4 antigens on the surface of hematopoietic stem cells is carried outin exactly the same fashion that is described for the first apsect ofthe invention. The step of administering a stimulating agent ofhematopoietic stem cell proliferation in vivo is preferably carried outthrough the administration of cytokines.

Preferred cytokines for stimulating hematopoietic stem cells toproliferate include granulocyte colony-stimulating factor (G-CSF), stemcell factor, totipotent stem cell factor (TSCF), stem cell proliferationfactor (SCPF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin,interleukin-1, -2, -3, -4, -6, and -11. Most preferred are G-CSF, stemcell factor and GM-CSF, because all three of these are known to causeproliferation of stem cells. The ability of G-CSF and GM-CSF tostimulate proliferation of progenitors is well established (see, e.g.,Metcalf, Nature 339:27-30 (1989)), as is their ability to causeperipheralization of hematopoietic stem cells (see, e.g., Haas et al.,Exp. Hematol. 18:94-98 (1990) and Blood 72:2074 (1988). This ability hasalso been established for stem cell factor (Andrews et al., Blood80:920-927 (1992)). In addition, the enormous potential of these othercytokines identified herein has been recognized (see Rowe and Rapoport,J. Clin. Pharmacol. 32:486-501 (1992)). For purposes of this invention,stimulation of hematopoietic stem cells to proliferate can be carriedout by any cytokine that is capable of mediating such proliferation invivo. Thus, for purposes of this invention, any cytokine that canstimulate hematopoietic stem cells to proliferate in vivo is consideredto be equivalent to G-CSF, stem cell factor and GM-CSF, which are alsoconsidered to be equivalent to each other. In addition, the use ofchemotherapeutic agents alone can lead to the peripheralization ofprogenitors. Such agents can also be combined with VLA-4 blocking agentsin the method according to the present invention.

In this method according to the second aspect of the invention,cytokines are preferably administered parenterally. The cytokines arepreferably administered as a sterile pharmaceutical compositioncontaining a pharmaceutically acceptable carrier, which may be any ofthe numerous well known carriers, such as water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, and the like, orcombinations thereof. Preferably, the cytokine, if G-CSF, will beadministered at a dose between about 1 μg/kg body weight/day and about50 μg/kg body weight/day, most preferably at about 10-15 μg/kg bodyweight/day. Most preferably, cytokines will be administered over acourse of from about four to about ten days. Optimization of dosages orthe combination of cytokines (e.g., G-CSF and kit ligand) can bedetermined by administration of the cytokine and administration of theblocking agents, followed by CFU-GM assay of peripheral blood. Thepreferred dosage should produce an increase of at least 5-fold in theCFU-GM counts per milliliter of peripheral blood, compared withcytokines alone.

According to this aspect of the present invention, the step ofadministering a blocking agent of VLA-4 antigens on the surface ofhematopoietic stem cells or CD34⁺ cells and the step of administeringstimulating agents for proliferation of these cells can be carried outconcomitantly or sequentially. In a preferred embodiment, the steps arecarried out sequentially, preferably administering stimulating agents ofCD34⁺ or hematopoietic stem cell proliferation being the first step.

In a third aspect, this invention provides an improved method oftransplanting peripheral blood stem cells into a patient who hasundergone chemotherapy or radiotherapy for cancer. In this method, priorto the administration of chemotherapy or radiotherapy, stem cells areperipheralized from the patient's bone marrow by administration of anagent that mediates blocking of VLA-4 antigens on the surface ofhematopoietic stem cells and CD34⁺ cells. The blocking agents used inthis method are preferably selected from those blocking agents describedin the discussion of the first aspect of the invention. This agent maybe administered alone, or in conjunction with an agent that stimulatesproliferation of stem cells. The proliferation stimulating agentsoptionally used in this method are preferably selected from thoseproliferation stimulating agents described in the discussion of thesecond aspect of the invention. The peripheralized stem cells are thencollected from peripheral blood by leukapheresis. Stem cells are thenenriched from the collected peripheralized blood by CD34 affinitychromatography such as immunoadsorption using anti-CD34 antibodies. Suchstem cell enrichment is known in the art and has been described, forexample, by Berenson, Transplantation Proceedings 24:3032-3034 (1992)and the references cited therein. Optionally, the enriched stem cellsare then expanded ex vivo by culturing them in the presence of agentsthat stimulate proliferation of stem cells. This ex vivo expansion canbe carried out using, alone or in combination, any of the proliferationstimulating agents described in the discussion of the second aspect ofthe invention. Such ex vivo expansion of CD34⁺ cells from peripheralblood is known in the art and has been described, for example, byBruggar et al., Blood 81:2579-2584 (1993). Following administration ofchemotherapy or radiotherapy, the enriched and optionally expanded stemcells are then returned to the patient's circulating blood and allowedto engraft themselves into the bone marrow.

The value of using peripheralized stem cells for transplantation afterchemotherapy or radiotherapy for cancer is recognized in the art and hasbeen described in numerous references, including Bensinger et al., Blood81:3158-3163 (1993); Chao et al., 81:2031-2035 (1993); Kessinger andArmitage, Blood 77:211-213 (1991); Gale et al., Bone MarrowTransplantation 9:151-155 (1992); and Siena et al., Blood 74:1904-1914(1989). The present method according to the invention provides animprovement in the transplantation of stem cells from peripheral bloodby increasing the concentration of such stem cells in the peripheralblood, thereby greatly improving the likelihood of success of thetransplantation.

In a fourth aspect, the present invention provides an improved method oftransplanting purified peripheral blood stem cells into a patient whohas undergone myeloablative chemotherapy or radiotherapy for AIDS. Thismethod involves the same steps as described for transplantingperipheralized stem cells into a patient who has undergone chemotherapyor radiotherapy for cancer. In addition, this method further optionallyinvolves administration to the patient of anti-HIV agents, such asantivirals such as AZT, soluble CD4, and CD4-directed blockers of theAIDS virus or antisense or antigene oligonucleotides, both before andafter the return of the enriched and optionally expanded stem cells tothe patient's circulating blood. This step serves a “mopping up”function to prevent residual virus from infecting the progeny of thenewly returned stem cells.

The myeloablative chemotherapy or radiotherapy will generally beexpected to destroy any cells in the blood that are infected by HIV. The“mopping up” step thus serves to remove any residual virus thatotherwise could possibly infect the progeny of the stem cellstransplanted into the patient after such therapy. Several agents can beuseful in such a “mopping up” step. For example, CD4-directed anti-HIVagents and analogs have been shown to prophylactically prevent infectionof uninfected CD34⁺ cells by HIV. Similarly, anti-HIV oligonucleotideshave been shown to prevent HIV infection of uninfected cells, forexample in U.S. Pat. No. 4,806,463, the teaching of which are herebyincorporated by reference. Such oligonucleotides have been shown toprevent virus escape for up to a 100 day test period. See Lisziewicz etal., Proc. Natl. Acad. Sci. U.S.A. 90:3860-3864 (1993). Accordingly,this method according to the invention should provide a new therapeuticapproach to AIDS.

In a fifth aspect, this invention provides an improved method forcarrying out gene therapy in patients having any of a variety of geneticand acquired diseases. In this method, stem cells are peripheralizedfrom the patient's bone marrow by administration of an agent thatmediates blocking of VLA-4 antigens on the surface of hematopoietic stemcells and CD34⁺ cells. The blocking agents used in this method arepreferably selected from those blocking agents described in thediscussion of the first aspect of the invention. As in the methodpreviously described herein, this agent may be administered alone or inconjunction with an agent that stimulates proliferation of stem cells.The proliferation stimulating agent optionally used in this method ispreferably selected from those proliferation stimulating agentsdescribed in the discussion of the second aspect of the invention.Peripheral blood is then collected by leukapheresis. Stem cells are thenenriched from the collected peripheral blood by immunoadsorption usinganti-CD34 antibodies. Such stem cell enrichment is known in the art andhas been described, for example, by Berenson, TransplantationProceedings 24:3032-3034 (1992) and the references cited therein.Optionally, the enriched stem cells are then expanded ex vivo byculturing them in the presence of agents that stimulate proliferation ofstem cells. This ex vivo expansion can be carried out using, alone or incombination, any of the proliferation stimulating agents described inthe discussion of the second aspect of the invention. Such ex vivoexpansion of CD34⁺ cells from peripheral blood is known in the art andhas been described, for example, by Bruggar et al., Blood 81:2579-2584(1993). The enriched and optionally expanded stem cells are theninfected with an amphotrophic retroviral vector, or other appropriatevector, that expresses a gene that ameliorates the genetic or acquireddisease. Optionally, the vector may also carry an expressed selectablemarker, in which case successfully transduced cells may be selected forthe presence of the selectable marker. The transduced and optionallyselected stem cells are then returned to the patient's circulating bloodand allowed to engraft themselves into the bone marrow. The usefulnessof approaches to using stem cells from peripheral blood forretroviral-mediated gene transfer and subsequent transplantation into apatient is recognized in the art and has been described, for example, byBragni et al., Blood 80:1418-1422 (1992). The present method accordingto the invention provides an improvement in the transplantation of stemcells from peripheral blood by increasing the concentration of such stemcells in the peripheral blood, thereby greatly improving the likelihoodof success of the retroviral transfection and subsequent transplantationand allows for repeated administration of genetically engineered cellsin patients with partially ablative regimens and receiving agents thatpromote proliferation of transduced cells. Such stem cell enrichment isknown in the art and has been described, for example, by Berenson,Transplantation Proceedings 24:3032-3034 (1992) and the references citedtherein.

The instant invention is useful for many purposes. The methods ofperipheralizing hematopoietic stem cells or CD34⁺ cells is of value inscientific research dedicated to understanding the molecularinteractions and molecular signals involved in the homing of these cellsto bone marrow, as well as their trafficking in response to certaininfections and trauma. This invention also provides sources ofperipheral blood that is enriched in CD34⁺ and hematopoietic stem cells,thus making the methods of the invention useful for therapeuticapplications involving autologous transplantation of these cell typesfollowing chemotherapy or radiotherapy or in the course of gene therapy.The present invention provides many advantages over the currentexclusively cytokine-based techniques. For example, peripheralizationcan be obtained without risk of cytokine-induced cell differentiation ofnormal cells or proliferation of contaminating leukemia cells and can becombined with cytotoxic agents. In addition, in the method of theinvention, the timing of the peak of progenitors in peripheral blood isconsistently between about 24 and about 72 hours from first injection ofantibody, thus making the most beneficial timing for leukapheresis morepredictable.

The efficacy of specific embodiments of methods according to bothaspects of the instant invention is demonstrated in the examples.According to the first aspect of the invention, monoclonal antibodiesagainst VLA-4 were administered to both macaques and a baboon. Theseantibodies, mouse monoclonal HP1/2, have previously been described byPulido et al., J. Biol. Chem. 266:10241 (1991), and are known to blockVLA-4 antigen on various cell surfaces. In the present case,administration of these antibodies resulted in as much as a 80-foldincrease (average of 40-fold) in CFU-GM present in peripheral blood. Thewell known CFU-GM assay is the most widely used measure of thehematopoietic progenitor viability of a PBSC harvest and correlates wellwith per cent CD34⁺ cells present in peripheral blood (see Craig et al.,Blood Reviews 6:59-67 (1992)). Thus, these results demonstrate that, ina primate, administering a blocking agent of VLA-4 antigen on thesurface of hematopoietic stem cells and CD34⁺ cells results inperipheralization of the hematopoietic stem cells and CD34⁺ cells. Theseresults should be applicable to humans as well.

According to the second aspect of the invention, monoclonal antibodiesagainst VLA-4 were administered to a macaque after five days oftreatment with G-CSF. It is well known that G-CSF can stimulatehematopoietic stem cells and CD34⁺ cells in vivo (see Metcalf, Nature339:27-30 (1989)). G-CSF alone caused an increase in CFU-GM present inperipheral blood by days 4 and 5 of treatment. After discontinuation ofG-CSF treatment and commencement of treatment with anti-VLA-4antibodies, the number of CFU-GM in peripheral blood increased even moredramatically. It will be recognized by those skilled in the art thatG-CSF alone does not cause the type of post-treatment increases inCFU-GM that were observed in the present case, as confirmed by a controlexperiment using G-CSF alone. Thus, these results demonstrate that, in aprimate, administering a blocking agent of VLA-4 antigen on the surfaceof hematopoietic stem cells and CD34⁺ cells in combination withadministering a stimulating agent for proliferation of these cells has asynergistic effect. There is no reason to believe that these resultswill not apply equally well to humans.

Although not wishing to be bound by theory, Applicant believes thatadministering a blocking agent of VLA-4 antigens on the surface ofhematopoietic stem cells and CD34⁺ cells causes peripheralization ofthese cells by mediating release of the cells from the marrowenvironment via disruption of interactions between VLA-4 and itsmicroenvironmental ligands, such as fibronectin and/or VCAM-1 on stromalcells or in the ECM. Administering stimulating agents of hematopoieticstem cell and CD34⁺ cell proliferation is believed to causeperipheralization at least in part via sheer increase in the numbers ofthese cells. Thus, it is believed that administering a blocking agent ofVLA-4 antigens in combination with a stimulating agent of stem cellproliferation effect peripheralization by complementary mechanisms. Theobserved synergistic effect between anti-VLA-4 antibodies and G-CSFsupports this interpretation. In addition, the observed synergisticeffect between anti-VLA-4 antibodies and 5-fluorouracil further confirmsthis interpretation. Since these mechanisms appear to be complementary,the observed synergistic effect should be observed, regardless ofwhether administration of the blocking agent of VLA-4 antigens andstimulation of proliferation are carried out concomitantly or insequence.

The following example are intended to further illustrate certainpreferred embodiments of the invention and are not intended to belimiting in nature.

EXAMPLE 1 Peripheralization Of Stem Cells Using An Anti-VLA-4 Antibody

Three macaques and one baboon were injected intravenously withanti-VLA-4 mouse monoclonal antibody HP1/2 (1 mg/kg body weight/day) forfour consecutive days. At various time points during and aftercompletion of treatment, peripheral blood was collected and mononuclearcells were collected using a conventional Ficoll-Hypaque separationprocedure. Total white blood cells were calculated from the number ofmononuclear cells recovered per milliliter of blood. CFU-GM and BFUewere determined according to conventional assays (see, e.g.,Papayannopoulou et al., Science 224:617 (1984)). The results of thesestudies are shown for two macaques (panels A and C) and one baboon(panel B) in FIG. 1. These results demonstrate that treatment of theseprimates with an anti-VLA-4 monoclonal antibody causes a small increase(up to 2-fold) in the total white blood cell count, peaking at about 2to 4 days after beginning of treatment. More importantly, the totalCFU-GM per ml blood increased much more dramatically (about 40-fold),also peaking at about 2 to 4 days after beginning of treatment. Inanother macaque, a CFU-GM increase of about 8-fold was observed after asingle injection of antibody (data not shown). Given the wellestablished use of the CFU-GM assay to measure the repopulatingpotential of hematopoietic progenitors and the correlation betweenCFU-GM and percentage CD34⁺, these results establish that the anti-VLA-4antibodies cause peripheralization of stem cells.

EXAMPLE 2 Failure of CD18 Blocking Agents to Cause Peripheralization ofStem Cells

The antigen CD18 is present on stem cells and is widely believed to beimportant in interactions involving stem cells. To test whether blockingagents for CD18 could cause peripheralization of stem cells, anothermacaque was treated with a monoclonal antibody against CD18. Antibodywas delivered by intravenous injection for three days at a dosage of 2mg/kg of body weight/day. The results of this control experiment areshown in FIG. 2. Total white blood cell counts did increase with thistreatment, consistent with previous experiments with rabbits. However,total GFU-GM showed no increase after treatment with anti-CD18monoclonal antibodies. Thus, even though CD18 is widely believed to beimportant in interactions involving stem cells, blocking agents of CD18do not lead to peripheralization of stem cells or progenitor cells.These results confirm that the peripheralization of stem cells observedupon treatment with anti-VLA-4 monoclonal antibody was indeed due tospecific blocking of VLA-4.

EXAMPLE 3 Synergistic Peripheralization Of Stem Cells Resulting FromTreatment With Both Anti-VLA-4 Antibody In Combination With G-CSF

A baboon was treated with recombinant human G-CSF twice daily for fiveconsecutive days. Each G-CSF treatment consisted of intravenousinjection of 15 micrograms G-CSF per kilogram of body weight. After thefive days of G-CSF administration, the baboon received two injections,spaced one day apart, of anti-VLA-4 monoclonal antibody (HP1/2). Eachinjection contained 1 milligram antibody per kilogram body weight. Totalwhite blood cells and CFU-GM were determined as described in Example 1.The results are shown in FIG. 3. As shown in panel A of that figure,G-CSF resulted in the expected increase in CFU-GM by days 4 and 5 oftreatment, along with a marked increase in total white blood cells.Surprisingly, after the administration of anti-VLA-4 antibody beginningafter the last day of a 5 day G-CSF treatment, yet another markedincreased in CFU-GM was observed, this time without any increase intotal white blood cells. This second increase resulted in about asix-fold improvement in the number of CFU-GM, relative to G-CSF alone. Acontrol animal treated with G-CSF alone according to the same protocolshowed a continuous decline in peripheral blood CFU after cessation oftreatment (see FIG. 3, panel B). These results indicate that treatmentwith anti-VLA-4 antibody was responsible for this second increase inCFU-GM. Thus, combined treatment with anti-VLA-4 antibody and G-CSFresults in a synergistic effect, causing far greater increases in CFU-GMthan treatment by either G-CSF or anti-VLA-4 antibodies alone.

EXAMPLE 4 Analysis Of High Proliferative Potential Cells In PeripheralBlood Following Combined Treatment With G-CSF And Anti-VLA-4 Antibody

In the experiments described in Example 3, high proliferative potential(HPP) cells were also counted. HPP cells are cells that give rise tocolonies that are macroscopically visible, over 0.5 mm in diameter withdense, compact growth on the analysis grid. Presence of these cells isassociated with greater repopulation capacity and such cells arebelieved to be earlier progenitors. The results are shown in FIG. 4. Theobserved disparity in peripheral blood HPP cells between G-CSF treatmentalone and G-CSF treatment in combination with anti-VLA-4 antibodies iseven greater than the disparity observed for CFU-GM. These resultssuggest that the combined treatment not only produces more progenitors,but also produces earlier progenitors having potentially greaterrepopulation capacity.

EXAMPLE 5 Synergistic Peripheralization Of Stem Cells Resulting FromTreatment With Anti-VLA-4 Antibody In Combination With 5-Fluorouracil

A baboon was treated with the chemotherapeutic agent 5-fluorouracil at adosage of 100 mg per kilogram body weight. Beginning five days later,the baboon received four injections, spaced one day apart, of anti-VLA-4monoclonal antibody (HP1/2). Each injection contained one milligramantibody per kilogram body weight. Total white blood cells and CFU-GMwere determined as described in Example 1. The results are shown in FIG.6. As shown in panel B of that figure, 5-fluorouracil alone produced amodest increase in CFU-GM at days 11 and 12. Administration ofanti-VLA-4 antibody after the 5-fluorouracil, however, resulted in adramatic further increase in CFU-GM, an increase of greater than tentimes that produced by 5-fluorouracil alone. These results indicate thatcombined treatment with anti-VLA-4 antibody and 5-fluorouracil producesa synergistic effect, causing far greater increases in CFU-GM thantreatment with either agent alone. Moreover, when taken together withthe G-CSF/anti-VLA-4 antibody results, these results strongly supportthe theory that the observed synergism results from stimulation ofproliferation of progenitors by one agent and release of the progenitorsfrom the marrow by another. Thus, these results strongly suggest thatsuch a synergistic effect can be produced by any agent that canstimulate proliferation, in conjunction with any agent that can bringabout release from the marrow.

EXAMPLE 6 Preparation Of A Humanized Anti-VLA-4 Antibody

The complementarity determining regions (CDRs) of the light and heavychains of the anti-VLA-4 monoclonal antibody HP1/2 were determinedaccording to the sequence alignment approach of Kabat et al., 1991, 5thEd., 4 vol., Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services, NIH, U.S.A. The CDRs of murineHP1/2 V_(H) correspond to the residues identified in the humanized V_(H)sequences disclosed herein as amino acids 31-35 (CDR1), 50-66 (CDR2) and99-110 (CDR3), which respectively correspond to amino acids 31-35, 50-65and 95-102 in the Kabat alignment. The CDRs of murine HP1/2 V_(K)correspond to the residues identified in the humanized V_(K) sequencesdisclosed herein as amino acids 24-34 (CDR1), 50-56 (CDR2) and 89-97(CDR3), and to the same residues in the Kabat alignment. The Kabat NEWMframework was chosen to accept the heavy chain CDRs and the Kabat REIframework was chosen to accept the kappa chain CDRs. Transplantation ofthe CDRs into the human frameworks was achieved by using M13 mutagenesisvectors and synthetic oligonucleotides containing the HP1/2 CDR-encodingsequences flanked by short sequences derived from the frameworks. TheV_(H) mutagenesis vector, M13VHPCR1 contains the NEWM framework and hasbeen described by Orlandi et al., Proc. Natl. Acad. Sci U.S.A.86:3833-3837 (1989). The V_(K) mutagenesis vector, M13VKPCR2 containsessentially the REI framework and is identical to the M13 VKPCR1 vectordescribed by Orlandi et al., except that there is a single amino acidchange from Val to Glu in framework 4. Transplanted product wasrecovered by PCR and cloned into M13mp19 for sequencing. Thetransplanted V_(H) sequence [SEQ. ID NO:3] is shown in FIG. 7, panel A.In addition to the CDR grafting, this product encodes the murine aminoacids at positions 27-30 and an Arg to Asp change at position 94. Thetransplanted V_(K) sequence [SEQ. ID NO:4] is shown in FIG. 7, panel B.

Additional modifications were introduced via the two step PCR-directedmutagenesis method of Ho et al., Gene 77:51-59 (1989). For the V_(H)sequence, position 24 (Kabat numbering) was changed from Val to Ala andposition 75 (Kabat numbering) was changed from Lys to Ser, then aminoacid positions 27-30 and 94 were mutated back to the NEWM sequences. Thefinal humanized V_(H) sequence [SEQ. ID NO:5] is shown in FIG. 8, panelA. For the V_(K) sequence, the same two step PCR-directed mutagenesisapproach was used to introduce additional modifications. The finalhumanized V_(K) sequence [SEQ. ID NO:6] is shown in FIG. 8, panel B.

The entire V_(H) and V_(K) regions of humanized HP1/2 were cloned intoappropriate expression vectors. The appropriate human IgG1, IgG4or kappaconstant region was then added to the vector in appropriate readingframe with respect to the murine variable regions. The vectors were thencotransduced into YB2/0 ray myeloma cells (available from ATCC), whichwere then selected for the presence of both vectors. ELISA analysis ofcell supernatants demonstrated that the humanized antibody produced bythese cells was at least equipotent with murine HP1/2. The cell lineexpressing this humanized antibody was deposited with the ATCC on Nov.3, 1992 and given accession number CRL 11175.

EXAMPLE 7 Peripheralization Of Stem Cells Resulting From Treatment WithHumanized Anti-VLA-4 Antibody

Humanized anti-VLA-4 antibodies prepared according to Example 6 weretested for peripheralizing stem cells. The baboon model was used againwith three daily antibody injections. The results are shown in FIG. 9.As previously shown for murine antibody, the humanized anti-VLA-4antibody produces a large increase in peripheralized CFU. Thus,humanized VLA-4 antibodies are capable of causing peripheralization ofstem cells and progenitor cells in the same manner as the murinemonoclonal antibody HP1/2. This result suggests that the humanizedantibody may also be capable, like the monoclonal antibody, of actingsynergistically in combination with G-CSF for peripheralizing stemcells.

EXAMPLE 8 Peripheralization Of Stem Cells Resulting From Treatment WithAnti-VLA-4 Murine Fab Fragment

Fab fragments from the murine antibody HP1/2 were tested for theirability to peripheralize stem cells and progenitor cells. The experimentwas performed by administration of 1 mg/kg of Fab fragment twice dailyfor three days. In this instance, a modest effect (compared withhumanized or monoclonal antibody) was observed, due to the rapidclearance of Fab fragments. Though modest, the observed characteristicBFU-e increase validates this result. This result demonstrates thatanti-VLA-4 antibody Fab fragments are capable of causingperipheralization of stem cells and progenitor cells. This suggests thatanti-VLA-4 Fab fragments may be capable of acting synergistically incombination with G-CSF for peripheralizing stem cells. In addition,since the Fab fragments are not known to have any effector functionother than binging antigen, this result suggests that any blocking agentthat can bind VLA-4 and thereby block its interaction with VCAM-1 willbe capable of peripheralizing stem cells, and in doing so, of actingsynergistically with factors that promote stem cell proliferation.

1. A method of peripheralizing CD34⁺ cells in vivo comprising the stepof administering an anti-VLA-4 antibody or an anti-VCAM-1 antibody whichblocks the binding of VLA-4 antigen on the surface of the CD34⁺ cells toVCAM VCAM- 1 or fibronectin, and thereby increasing the number of CD34 ⁺cells in the peripheral blood.
 2. The method according to claim 1,wherein the anti-VLA-4 or anti-VCAM-1 antibody is a mouse/human,chimeric antibody, single chain antibody, or humanized antibody, or Fab,Fab′, F(ab′)₂ or F(v) fragments thereof.
 3. The method according toclaim 1, wherein at least a portion of the CD34⁺ cells are hematopoieticstem cells.
 4. The method of claim 1, further comprising the step ofadministering a stimulating agent of CD34⁺ cell proliferation in vivo,said stimulating agent being 5-fluorouracil or a cytokine thatstimulates hematopoietic cells to proliferated proliferate.
 5. Themethod according to claim 2, further comprising the step ofadministering a stimulating agent of CD34⁺ cell proliferation in vivo,said stimulating agent being 5-fluorouracil or a cytokine thatstimulates hematopoietic cells to proliferated proliferate.
 6. Themethod according to claim 3, further comprising the step ofadministering a stimulating agent of hematopoietic stem cellproliferation in vivo, said stimulating agent being 5-fluorouracil or acytokine that stimulates hematopoietic cells to proliferatedproliferate.
 7. The method according to claim 4, wherein the stimulationis mediated by a cytokine selected from the group consisting ofgranulocyte colony-stimulating factor (G-CSF), stem cell factor,granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophagecolony-stimulating factor (M-CSF), totipotent stem cell factor (T-SCF),stem cell proliferation factor (SCPF), interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-6 (IL-6) and interleukin-11 (IL-11).
 8. The method accordingto claim 5, wherein the stimulation is mediated by a cytokine selectedfrom the group consisting of granulocyte colony-stimulating factor(G-CSF), stem cell factor, granulocyte-macrophage colony-stimulatingfactor (GM-CSF), macrophage colony-stimulating factor (M-CSF),totipotent stem cell factor (T-SCF), stem cell proliferation factor(SCPF), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3(IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-11(IL-11).
 9. The method according to claim 6, wherein the stimulation ismediated by a cytokine selected from the group consisting of granulocytecolony-stimulating factor (G-CSF), stem cell factor,granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophagecolony-stimulating factor (M-CSF), totipotent stem cell factor (T-SCF),stem cell proliferation factor (SCPF), interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-6 (IL-6) and interleukin-11 (IL-11).
 10. The methodaccording to claim 7, wherein the cytokine is G-CSF.
 11. The methodaccording to claim 8, wherein the cytokine is G-CSF.
 12. The methodaccording to claim 9, wherein the cytokine is G-CSF.
 13. The methodaccording to claim 10, wherein the G-CSF is administered beforeadministering the anti-VLA-4 antibody or anti-VCAM-1 antibody .
 14. Themethod according to claim 11, wherein the G-CSF is administered beforeadministering the anti-VLA-4 antibody or anti-VCAM-1 antibody .